It is challenging to develop anti-thrombotic therapeutics to treat or prevent pathological thrombus formation without increasing the risk of bleeding. Currently available anti-coagulant drugs render blood thinner irrespective of whether an inflammatory pro-thrombotic condition exists or whether clotting needs to occur because of traumatic vessel rupture. It is desirable to develop a drug that is active only under the oxidizing conditions present during inflammation. The blood protein von Willebrand factor (VWF) plays a key role in initiating blood clotting and it has been the target of anti-thrombotic therapies. It has been shown that oxidizing agents released during inflammation activate VWF by converting methionine residues within its domains to methionine sulfoxide. A previous study by us developed a method to computationally screen for drugs that inhibit VWF more strongly in the presence of oxidizing conditions. The computations suggested in particular a drug, lumacaftor, that could inhibit VWF function selectively in the presence of oxidized methionine residues. Here, we developed an enzyme-linked immunosorbent assay to test the effect of drugs on the ability of VWF to bind to the platelet surface receptor glycoprotein Ibα comparing oxidizing and non-oxidizing conditions. The results indicate that lumacaftor may indeed have the desired properties of inhibiting VWF selectively when oxidized. Because of its simplicity, the assay provides a high-throughput method to efficiently screen multiple drugs against VWF in both its oxidized and unoxidized state.

Endothelial dysfunction in patients after severe injury with hemorrhagic shock is associated with excessive release of ultralarge von Willebrand factor (VWF) without a corresponding increase in ADAMTS13 (A disintegrin and metalloprotease thrombospondin type 1 motifs, member 13), resulting in acquired ADAMTS13 deficiency and VWF hyperadhesive activity. We hypothesized that the VWF to ADAMTS13 imbalance is exacerbated by increased VWF oxidation, rendering it resistant to cleavage by ADAMTS13. Plasma indices of endotheliopathy and coagulopathy in 100 hemorrhagic shock patients were obtained, ADAMTS13-to-VWF ratio was calculated and correlated with indices of adverse outcomes using Classification and Regression Tree modeling. A novel mass spectrometry method was used to quantify the rate of VWF cleavage in individual patients. In a mouse model of polytrauma and hemorrhagic shock, a single dose of human recombinant ADAMTS13 was administered and VWF cleavage, VWF activity, and parameters of lung dysfunction examined. We detected a reduced rate of cleavage and increased oxidation rate of selective methionine residues of VWF in these patients. In mice, recombinant ADAMTS13 decreased the adhesive activity and enhanced cleavage of plasma VWF, and improved parameters of lung dysfunction. The present study supports the conclusion that an imbalance between VWF and ADAMTS13 drives, at least in part, the endotheliopathy of trauma and the associated adverse outcomes and suggests a potential therapeutic target for severely injured patients with hemorrhagic shock.

Traumatic brain injury (TBI) causes endothelial injuries both at the site of injury and in remote regions of the brain. TBI-induced endotheliopathy also disseminates to extracranial organs, such as the lungs. This TBI-induced systemic endotheliopathy has been extensively reported in clinical studies, but its underlying mechanism remains poorly understood. Here we report results from a study designed to investigate the role of extracellular mitochondria (exMt) in TBI-induced endotheliopathy. We showed that exMt are released from injured brains into the circulation, reaching a maximal level of 1.8x104/µl at 3 hours after mice were subjected to severe TBI. These exMt express peroxidized cardiolipin on their surface and generate significant amounts of reactive oxygen species, but they produce less ATP compared to intracellular mitochondria of normal cells. When infused into non-injured mice, exMt induce systemic endotheliopathy defined by increased endothelial permeability, tissue edema, and perivascular bleeding. We further demonstrated that endothelial cells endocytose exMt through the lipid-scavenging receptor CD36 and dynamin-driven clathrin-mediated pathway. The endocytosed exMt interacted with the endoplasmic reticulum (ER) of ECs through newly formed mitochondria-associated membrane, leading to intracellular ER stress and resultant apoptosis. This study delineates a new pathway of exMt-induced endotheliopathy during acute TBI. It also demonstrates a causal role of exMt in endothelial injuries associated with (poly)trauma, severe infection, and autoimmune diseases.

Background and Objective Exercise is known to reduce thrombotic risk and systemic inflammation, but the underlying mechanisms are not understood. One potential mechanism involves the exercise-induced myokine irisin, which is cleaved from fibronectin type III domain-containing protein 5 (FNDC5) during muscle contraction and has well-known metabolic benefits. Irisin's amino acid sequence is fully conserved across mammals, suggesting evolutionarily preserved function. The possible cardiovascular effects of irisin remain largely unexplored as irisin's receptors beyond the skeletal αVβ5 are unknown. In this study, we aimed to investigate the anti-thrombotic effects of irisin and identify its cellular targets and mechanisms of action. Methods Whole blood, platelet-rich plasma (PRP), washed platelets, and monocytes were obtained from healthy donors (n=25). Recombinant irisin was purified from transfected HEK293 cells and fluorescently labeled. Binding studies were performed on platelets, monocytes, and human umbilical vein endothelial cells (HUVECs) with flow cytometry under various conditions, including Mn²⁺ supplementation and temperature variations. Crosslinking was performed with DTSSP and proteomics with LC-MS/MS. Functional assays included measurement of platelet aggregation by light transmission aggregometry, platelet spreading by F-actin imaging, clot contraction by serial imaging, and expression of activation markers (CD62P and PAC-1 binding) after stimulation with collagen, convulxin, thrombin, arachidonic acid, or ADP, monocyte activation marker CD64, and PMA-induced von Willebrand factor (VWF) secretion in endothelialized microchannels. Thrombus formation in vivo was induced by laser-mediated injury of cremaster arterioles and monitored by intravital microscopy. Results Irisin demonstrated differential binding affinity across cell types, with endothelial cells showing the highest affinity, followed by monocytes (Kd ~30 nM, comparable to αVβ5 integrin), then platelets. Overall, irisin binding was enhanced by integrin activation (induced by Mn²⁺), cell activation, or warmer temperatures. In platelets, irisin binding was competitively inhibited by fibrinogen (p<0.0001) or PAC-1 (p<0.0001), while crosslinking and proteomics indicated αIIbβ3 integrin as the receptor, suggesting that irisin preferentially binds an active, open conformation of αIIbβ3. In platelets, irisin binding decreased CD62P exposure (p=0.04) especially at lower agonist concentrations, inhibited collagen- or convulxin-induced aggregation, and reduced thrombin-induced platelet spreading on fibrinogen-coated surface (p=0.03) without affecting overall clot contraction. For collagen, irisin inhibited aggregation dose-dependently: 250 nM irisin 54.7±19.3% vs control (p=0.12), 500 nM 35.9±24.2% (p=0.004), and 1µM 13.4±14.9% (p=0.0001). We obtained similar results for convulxin, but not with any other agonists tested. In whole blood, irisin inhibited shear-induced thrombus formation on collagen surfaces measured by T-TAS (50±16% vs control, p=0.32, for 1µM irisin and 6±6%, p=0.005, 2µM). Together, irisin inhibition of αIIbβ3seems to require a longer lag time of inside-out agonist activation as provided by the GPVI agonists. In monocytes, irisin binding was enhanced by PMA-induced activation, resulted in a slight decrease in activation marker CD64, and was abolished by pretreatment with CBRM1/5 (binds active αM), suggesting that αMβ2 is the receptor. In endothelial cells, irisin bound more robustly to thrombin- and PMA-activated HUVECs, and reduced PMA-induced VWF secretion by 48±11.8% (p=0.004, 0.5µM). Finally, our preliminary study in wild-type mice showed that irisin pretreatment decreased platelet recruitment and unstable thrombus formation after laser-induced arteriolar injury. Conclusions: Irisin functions as a multi-target anti-thrombotic myokine that concertedly modulates platelet, monocyte, and endothelial cell function through distinct integrin receptors. By binding to activated or partially active αIIbβ3 on platelets, putatively αMβ2 on monocytes, and likely an αV integrin on endothelial cells, irisin either blocks the intermediate conformations of the integrins or competes with physiological pro-thrombotic ligands resulting in both anti-thrombotic and anti-inflammatory effects. This coordinated multi-cellular modulation positions irisin as an endogenous, exercise-inducible therapy for thrombotic disorders.

ABSTRACT Background Platelets are stored at room temperature for 5-7 days (RSP). Due to frequent and severe shortages, the FDA recently approved up to 14-day cold-stored platelets in plasma (CSP). However, the post-transfusion function of CSP is unknown and it is unclear which donors are best suited to provide either RSP and/or CSP. Objective To evaluate the post-transfusion function and predictors of post-transfusion function for platelets stored for the maximum approved storage times (7-day RSP, 14-day CSP) in healthy volunteers on acetylsalicylic acid (ASA). Methods We conducted a randomized cross-over study in ten healthy humans. Subjects donated one platelet unit stored at either RT (RSP) or 4 °C (CSP) based on randomization. Before transfusion, subjects ingested ASA to inhibit endogenous platelets. Transfusion recipients were tested for platelet function and lipid mediators. Platelet units were tested for lipid mediators only. A second round with transfusion of the alternative product and an identical testing sequence followed. Results RSP reversed platelet inhibition significantly better in αIIbβ3 integrin activation-dependent assays. In contrast, CSP led to significantly more thrombin generation in recipients, which was not dependent on platelet microparticles, but CSP themselves. Lysophosphatidylcholine-O (Lyso-Platelet Activating Factor) species levels predicted the procoagulant capacity of CSP. In contrast, polyunsaturated fatty acid concentration predicted the aggregation response of RSP. Conclusion We provide the first efficacy data of extended-stored CSP in plasma. Our results suggest that identifying ideal RSP and CSP donors is possible and pave the way for larger studies in the future. Graphical Abstract 1: Overview of CSP function after 14 days of storage (Created with Biorender)

Background: Platelets are stored at room temperature for 5-7 days (RSP) and are transfused to patients who are bleeding or at risk of bleeding. Due to frequent and severe shortages, the FDA recently approved cold-stored platelets in plasma (CSP) for up to 14 days despite a lack of post-transfusion data. It is also unclear which donors are best suited to provide platelets for either RSP or CSP and which in vitro markers accurately predict post-transfusion RSP or CSP platelet function. Objective: To evaluate the post-transfusion function and predictors of post-transfusion function of platelets stored for the maximum approved storage times (7-day RSP, 14-day CSP) in healthy volunteers on acetylsalicylic acid (ASA). Methods: We conducted a randomized cross-over study in ten healthy humans. Subjects on ASA were randomized to receive either autologous RSP or CSP first. We obtained blood from recipients before and after transfusion for platelet function testing and samples from the storage bag before and after storage for lipid metabolite analysis. The first round was followed by a second round with transfusion of the alternative product and the same testing sequence. Results: RSP reversed platelet inhibition significantly better in αIIbβ3 integrin activation-dependent assays. In contrast, CSP led to significantly more thrombin generation in recipients. Overall, RSP contained more pro-inflammatory lipid mediators while CSP had more anti-inflammatory lipid mediators. In particular, LPC species including LPC-O species (formerly known as Lyso-platelet activating factor [LPAF]), LPE, and LPI were significantly more abundant in RSP than in CSP, while LPS was more abundant in CSP than in RSP. Similarly, the relative increase of LPC, LPE, and LPI, and linoleic acid-dependent oxylipins, such as diHOMEs and HODEs were higher in RSP recipients than in CSP recipients. Linoleic acid-dependent oxylipins correlated positively with platelet aggregation in units and recipients of RSP and CSP. Polyunsaturated fatty acids (PUFAs) showed a negative correlation with platelet aggregation in both RSP and CSP units and recipients. Most importantly, LPC-O (LPAF) species showed a strong negative correlation with endogenous thrombin generation potential in CSP units after collection (pre-storage), post-storage, and in transfusion recipients. Conclusion: Results from our trial provide the first efficacy data of autologous and extended-stored CSP in plasma. We report the first correlational data between unit and recipient lipid mediators and post-transfusion platelet function. Taken together, we show that the main mechanism of post-transfusion CSP function appears to be based on procoagulant properties. In the future, testing platelet donors for LPC-O (LPAF) species could identify donors whose platelets are better suited for cold storage than room-temperature storage.

von Willebrand factor (VWF) mediates primary hemostasis and thrombosis in response to hydrodynamic forces. We previously showed that high shear promoted self-association of VWF into hyperadhesive strands, which can be attenuated by high-density lipoprotein (HDL) and apolipoprotein A-I. In this study, we show that low-density lipoprotein (LDL) binds VWF under shear and enhances self-association. Vortexing VWF in tubes resulted in its loss from the solution and deposition onto tube surfaces, which was prevented by HDL. At a stabilizing HDL concentration of 1.2 mg/mL, increasing concentrations of LDL progressively increased VWF loss, the effect correlating with the LDL-to-HDL ratio and not the absolute concentration of the lipoproteins. Similarly, HDL diminished deposition of VWF in a post-in-channel microfluidic device, whereas LDL increased both the rate and extent of strand deposition, with both purified VWF and plasma. Hypercholesterolemic human plasma also displayed accelerated VWF accumulation in the microfluidic device. The initial rate of accumulation correlated linearly with the LDL-to-HDL ratio. In Adamts13-/- and Adamts13-/-LDLR-/- mice, high LDL levels enhanced VWF and platelet adhesion to the myocardial microvasculature, reducing cardiac perfusion, impairing systolic function, and producing early signs of cardiomyopathy. In wild-type mice, high plasma LDL concentrations also increased the size and persistence of VWF-platelet thrombi in ionophore-treated mesenteric microvessels, exceeding the accumulation seen in similarly treated ADAMTS13-deficient mice that did not receive LDL infusion. We propose that targeting the interaction of VWF with itself and with LDL may improve the course of thrombotic microangiopathies, atherosclerosis, and other disorders with defective microvascular circulation.

We recently demonstrated that plasma lipoproteins play a major role in regulating the function of the hemostatic system by either inhibiting or promoting the self-association of von Willebrand factor (VWF) into large fibers and bundles that are hyperadhesive for platelets. Such fibers can form on the surface of activated endothelium such as during thrombotic microangiopathies. We showed that high-density lipoprotein (HDL) attenuates VWF self-association and reduces the formation of VWF-platelet thrombi, both in vitro and in mice (PMID:26552698). By contrast, low-density lipoprotein (LDL) enhances VWF self-association and promotes the formation of VWF-platelet thrombi ( Blood, in press). Consequently, wild-type mice with elevated plasma LDL display intense and prolonged VWF-platelet thrombosis in stimulated mesenteric microvessels, exceeding even the thrombosis observed in ADAMTS13-deficient mice with normal LDL levels. All the lipoproteins of low density carry apolipoprotein (apo) B100 and comprise fractions of very low density (VLDL), intermediate density (IDL), and low density (LDL). The LDL fraction comprises two subfractions: small-dense (sd) LDL and large-buoyant (lb) LDL. Epidemiologic studies suggest that sd-LDL is the most atherogenic subfraction (PMID:33586462). Another atherogenic lipoprotein also carries apoB-100, Lp(a), in which the apoB-100 is complexed with apo(a). Here, we sought to determine the effect of LDL subfraction(s) on VWF self-association. We isolated LDL, sd-LDL and lb-LDL from healthy donor plasma by sequential KBr density centrifugation. Lp(a) was purified from patients with high circulating Lp(a) (>50 mg/dL) as described by Mueller et al. (PMID:35650291). We first tested these lipoproteins in microfluidic devices adapted from the design of Herbig and Diamond (PMID:26178390). In these devices, high shear stress and flow acceleration are generated around a 30-um-wide pillar within a 60-um-wide flow channel and induce the attachment and self-association of VWF into fibers on and around the pillar. We quantified VWF fiber accumulation from differential interference contrast (DIC) images. Of all the lipoprotein fractions, sd-LDL was the most potent in accelerating VWF strand accumulation (see figure). The effect of lipoproteins in this device correlated with their impact on microvascular thrombosis in vivo. We infused apoB100-containing lipoproteins into wild-type mice (3-4 weeks old) before inducing VWF-platelet thrombi deposition in the mesenteric microvasculature with ionophore. All the apoB100 lipoproteins enhanced microvascular thrombosis [we did not test Lp(a), which had no effect in the microfluidic channel], but sd-LDL was by far the most potent; the treated mice had twice the number of large thrombi (>30-um diameter) as the mice treated with an equivalent quantity of lb-LDL. The effect also persisted for much longer before returning to baseline with the sd-LDL. We have explored possible reasons why sd-LDL is so much more potent. Compared to the other apoB-containing lipoproteins, sd-LDL is smaller and denser, with a greater surface curvature. Whether this fact changes accessibility to sites on apoB100 is not known. We also compared the lipidomes and proteomes of sd-LDL and lb-LDL for clues to the difference. Compared to lb-LDL, sd-LDL contained a much higher content of cholesterol and cholesterol esters per particle. The proteomes of the two particles were also different, with several proteins being more abundant in lb-LDL, in particular apoC-III, apoE, apoA-IV, and apoC-II. Only apoF was significantly more abundant in sd-LDL. We are currently testing to determine if any of these difference accounts for the differences between the two LDL subfractions in promoting VWF self-association. In summary, we show that the lipoprotein associated with the greatest atherosclerotic burden and most cardiovascular disease is also the one with the most potent effect in promoting a prothrombotic state by enhancing VWF self-association. These findings have important implications for the development of the atherosclerotic lesion, the formation of the thrombus after the lesion ruptures, and for the microvascular dysfunction often observed after revascularization.

Bleeding associated with left ventricular assist device (LVAD) implantation has been attributed to the loss of large von Willebrand factor (VWF) multimers to excessive cleavage by ADAMTS-13, but this mechanism is not fully supported by the current evidence. We analyzed VWF reactivity in longitudinal samples from LVAD patients and studied normal VWF and platelets exposed to high shear stress to show that VWF became hyperadhesive in LVAD patients to induce platelet microvesiculation. Platelet microvesicles activated endothelial cells, induced vascular permeability, and promoted angiogenesis in a VWF-dependent manner. Our findings suggest that LVAD-driven high shear stress primarily activates VWF, rather than inducing cleavage in the majority of patients.

AIM To investigate the effect of leptin on the angiogenesis of RF/6A cells (monkey retinal choroidal endothelial cells) in vitro and test the cellular signaling in the mechanism. METHODS RF/6A cells were cultured in vitro and randomly divided into four groups: normal control, with leptin at 50, 100, 200 ng/mL for cell counting kit-8 (CCK8). RF/6A cell proliferation and migration were examined by Transwell assays, while RF/6A cell tube formation by Matrigel assay. JAK2, p-JAK2, STAT3, and p-STAT3 protein expression was measured by Western blotting. Cells were then divided into the following treatment groups: control, 100 ng/mL leptin and AG-490 (100 ng/mL leptin+10 µmol/L AG-490) for examinations of RF/6A cellular behaviour again. Analysis of differences was carried out using one-way ANOVA and least significant difference (LSD). RESULTS RF/6A cell proliferation, migration and cell tube formation were promoted significantly by leptin in a dose-dependent manner (P<0.05). Western blotting showed that leptin up-regulated p-JAK2 and p-STAT3 expression levels. Treatment with the JAK/STAT pathway inhibitor, AG-490, decreased leptin-induced p-JAK2 and p-STAT3 expression, and inhibited cell proliferation, migration and cell tube formation induced by leptin (P<0.05). CONCLUSION Leptin can promote RF/6A cell angiogenesis in vitro via activation of the JAK2/STAT3 signaling pathway.

Homozygous familial hypercholesterolemia (HoFH) is a rare autosomal recessive genetic disorder. It is difficult to diagnose and treat it at early stage. We present a nine-year-old boy with HoFH from China. At the beginning, he was misdiagnosed as xanthomatosis in the dermatology department of the local hospital, but the disease did not alleviate after three laser ablation operations. Later, blood lipid monitoring, ultrasound of heart and carotid artery were further added in our hospital, and finally the boy was diagnosed with HoFH by genetic testing. A biallelic mutations was observed in the fourth exon of low density lipoprotein receptor (LDLR): c.418G>A (p.E140K). Our patient achieved a relatively satisfactory therapeutic results after a series of lipid-lowering therapies including atorvastatin monotherapy, lipoprotein apheresis and double-filtration plasma pheresis. We found that LDL-C levels obtained 57% reduction from baseline after atorvastatin combined with double-filtration plasma pheresis (DFPP). It was observed that regression of carotid intima-media thickness (cIMT), valve regurgitation and xanthoma occurred after a series of Intensive lipid-lowering therapy.

Platelets are currently stored at room temperature before transfusion to maximize circulation time. This approach has numerous downsides, including limited storage duration, bacterial growth risk, and increased costs. Cold storage could alleviate these problems. However, the functional consequences of cold exposure for platelets are poorly understood. In the present study, we compared the function of cold-stored platelets (CSP) and room temperature-stored platelets (RSP) in vitro, in vivo, and post-transfusion. CSP formed larger aggregates under in vitro shear while generating similar contractile forces compared to RSP. We found significantly reduced GPVI levels after cold exposure of 5-7 days. After transfusion in humans, CSP were mostly equivalent to RSP yet aggregated significantly less to the GPVI agonist collagen. In a mouse model of platelet transfusion, we found a significantly lower response to the GPVI-dependent agonist convulxin and significantly lower GPVI levels on the surface of transfused platelets after cold storage. In summary, our data support an immediate but short-lived benefit of CSP and highlight the need for thorough investigations of this product. (NCT03787927).

Visfatin is a type of adipocytokine that is highly expressed in the serum and vitreous of patients with diabetic retinopathy. The purpose of the present study was to investigate the effect and mechanism of visfatin on angiogenesis in RF/6A monkey chorioretinal retinal endothelial cells under high glucose (HG) conditions in vitro. RF/6A cells were randomly divided into four groups: Control group, under high glucose (HG) group (25 mM D-glucose), visfatin group 1 (10 nM visfatin + 25 mM D-glucose), visfatin group 2 (20 nM visfatin + 25 mM D-glucose) and visfatin group 3 (30 nM visfatin + 25 mM D-glucose). After 24 and 48 h, a Cell Counting Kit-8, wound-healing assay and Matrigel tube formation assay were used to detect cell proliferation, migration and cell tube formation, respectively. Subsequently, the expression levels of VEGF and VEGF receptor 2 (VEGFR-2) in cells of visfatin group 3 were observed by western blot and reverse transcription-quantitative PCR analyses. At 24 and 48 h, the cell proliferation and migration distance in the HG group were reduced compared with those in the control group (P<0.05). Compared with those in the HG group, the cell proliferation and migration distance in all visfatin groups were significantly increased (P<0.05), with the highest significance in visfatin group 3. Visfatin significantly promoted tube-like structure formation by RF/6A cells, particularly at the concentration of 30 nM. The protein and mRNA expression levels of VEGF and VEGFR-2 were significantly increased in the HG group as compared with those in the control group (P<0.05). Furthermore, compared with those in the HG group, VEGF and VEGFR-2 protein and mRNA expression levels were significantly increased in visfatin group 3 (P<0.05). Overall, visfatin promoted the proliferation, migration and tube formation of RF/6A cells under HG conditions, suggesting that visfatin has a potent effect on retinal neovascularization and its mechanism may be associated with the promotion of VEGF and VEGFR-2 expression under HG conditions.

Background. Coagulopathic bleeding is a major cause of mortality after trauma, and platelet dysfunction contributes to this problem. The causes of platelet dysfunction are unknown, but a great deal can be learned from the plasma environment after injury, which may directly alter platelet function. Studying the changes in plasma using untargeted proteomics would provide unbiased insight into the presence of possible inhibitors of platelet function, changes to their major ligands, or other previously unknown pathways affecting platelet function. Methods. Citrated blood was collected from severely injured trauma patients at the time of their arrival to the Emergency Department. Platelet testing was performed immediately, and plasma was frozen for analysis. Samples were collected from 110 patients, and a subset of 24 patients was identified by a preserved (n=12) or severely impaired (n=12) platelet aggregation response to five different agonists (adenosine diphosphate, arachidonic acid, collagen, thrombin receptor-activating peptide, and ristocetin). Untargeted proteomics was performed by nanoflow liquid chromatography tandem mass spectrometry to determine the plasma protein profile associated with platelet dysfunction. Protein abundance levels for each patient were normalized to total protein concentration to control for hemodilution by crystalloid fluid infusion prior to blood draw. Results were compared by Wilcoxon rank-sum test, and a two-tailed p value less than 0.05 was considered significant. No adjustment was made for multiple comparisons, as the risk of a type II error was felt to outweigh that of a type I error in this exploratory analysis. Results. Patients with platelet dysfunction were more severely injured (median Injury Severity Score 29.5 vs. 13.5, p=0.002) but otherwise demographically similar to those with retained platelet function. Of 232 proteins identified, twelve were significantly different in the low- vs. high-platelet function groups: gelsolin (median peak intensity 9.70E+6 vs. 1.48 E+7, p=0.002), transketolase (2.86E+4 vs. 9.36E+3, p=0.003), protein S100-A8 (4.60E+4 vs. 2.64E+4, p=0.006) and -A9 (5.83E+4 vs. 4.07E+4, p=0.012), histone H4 (5.72E+4 vs. 8.47E+6, p=0.007), histidine-rich glycoprotein (HRG) (5.00E+6 vs. 8.30E+6, p=0.008), factor XIII B chain (1.09E+6 vs. 1.61E+6, p=0.020), apolipoprotein A-IV (2.30E+7 vs. 3.28E+7, p=0.024), alpha-enolase (1.02E+5 vs. 4.15E+4, p=0.024), heat shock protein 90-alpha (1.41E+4 vs. 4.08E+3, p=0.045), alpha-2-HS-glycoprotein (2.83E+7 vs. 3.68E+7, p=0.045), and neutrophil gelatinase-associated lipocalin (3.44E+4 vs. 1.36E+4, p=0.045). These results are summarized in the figure, which shows one group of proteins that decreased in abundance (on the left side of the volcano plot) and another that increased (on the right side) in the patients with low platelet function. The twelve proteins that changed the most fall into several categories related to platelet function. Low gelsolin and high histone levels are each associated with microvascular obstruction by release of intracellular material that accumulates in small vessels and activates platelets. Low HRG and high damage-associated molecular pattern (DAMP) protein levels are consistent with massive innate immune activation, which can impair platelet function in many ways. Conclusion. This study provides an unbiased description of the change in proteomic profile associated with platelet dysfunction after trauma and identifies twelve proteins with the most profound changes. The pathways involving these proteins are salient targets for immediate investigation to better understand platelet dysfunction after trauma and identify targets for intervention. Figure Legend. Volcano plot depicting fold-change and statistical significance of individual proteins in patients with low vs. high platelet function. Dotted horizontal line represents p = 0.05. Twelve proteins showed a statistically significant p value. Figure Disclosures No relevant conflicts of interest to declare.

Introduction: Choroidal neovascularization (CNV) is the main pathological change of wet age-related macular degeneration. Anti-VEGF drugs are the most commonly used treatment for CNV. The biggest drawback of antiVEGF drugs is the recurrence of CNV, which requires repeated therapy several times. Autophagy activation may be involved in reducing the therapeutic effect of anti-VEGF drugs. So, this study aims to elucidate the effect and mechanism of anti-VEGF drugs on endothelial autophagy and neovascularization in vitro. Methods: RF/6A cells were randomly divided into five groups: The control group, hypoxia group (1% O2, 5% CO2, 94% N2), anti-VEGF group (group1: Ranibizumab 100 mu g/ml; group2: Aflibercept, 400 mu g/ml; group3: Conbercept, 100 mu g/ml). Autophagy-related proteins were examined by Western blot. RFP-GFP-LC3 was used to detect autophagy and autophagic flow. Subsequently, we used autophagy inhibitors (3-MA or CQ) to inhibit Conbercept induced autophagy and to observe its effect on angiogenesis in vitro. Proliferation, migration, and tube formation of endothelial cells can be used to study neovascularization in vitro. In this research, the CCK-8 assay was used to detect cell proliferation. Cell migration and tube formation were assessed by wound assay and matrix method, respectively. Flow cytometry and Tunel were used to detect cell apoptosis. Finally, the mechanism of Conbercept activated autophagy was studied. Western blot was used to detect the expression of p53 and DRAM (damage-regulated autophagy modulator), upstream activators of autophagy. Results: The protein levels of Beclin-1 and LC3-2/1 in Ranibizumab and Conbercept groups were significantly higher than in the hypoxia group(P < 0.05). While the expression of P62 was decreased (P < 0.05). The autophagic flux was showed the same results. However, Aflibercept showed the opposite effect on autophagy. Compared with the Conbercept group, autophagy inhibitor 3-MA or CQ can further inhibit cell proliferation and promotes cell apoptosis (P < 0.05). Conbercept significantly inhibited cell migration compared with the hypoxia group (633.08 +/- 72.52 vs. 546.33 +/- 24.61), while the autophagy inhibitor group (3-MA or CQ) had a more obvious inhibition effect (309.75 +/- 86.36 and 263.33 +/- 68.67) (P < 0.05). For tube formation, the number of tube formation was decreased significantly in the Conbercept group (32.00 +/- 2.00) compared to the hypoxia group (39.00 +/- 1.53) and even further reduced in 3-MA or CQ group (24.00 +/- 3.61, 20.00 +/- 2.65). The length of master segments in the hypoxia group was 15,668.00 +/- 894.11. It was decreased in Conbercept (13,885.34 +/- 730.03). In 3-MA or CQ group, the length of master segments dropped further (11,997.00 +/- 433.66, 10,617.67 +/- 543.21). Compare with the hypoxia group, the expression P53 and DRAM were increased in the Conbercept group (P < 0.05). Autophagy-related proteins LC-3, Beclin-1, and DRAM were inhibited by P53 inhibitor Pifithrin-alpha (PFT alpha) (P < 0.05). Conclusion: Ranibizumab and Conbercept can trigger the autophagy of vascular endothelial cells while Aflibercept can inhibit it. The combination of Conbercept and autophagy inhibitor can significantly inhibit the formation of angiogenesis in vitro. The mechanism of autophagy activation is related to the activation of the p53/DRAM pathway.

Background: Platelet transfusion is a potentially lifesaving procedure, used for both prophylactic and therapeutic indications. Platelets can be stored at room temperature (RT) for up to 7 days in air-permeable bags. Platelet function diminishes during storage, a phenomenon known as the storage lesion. We and others have shown that platelets can be stored for extended periods of time at 4°C and still show acceptable in vitro function while limiting bacterial growth. In the present study, we used proteomics to examine the changes in human platelets stored at RT and 4°C with a focus on the glycoprotein (GP) Ib-IX-V complex, the key receptor for platelet adhesion at sites of vessel injury. Study Design/Method: Platelet units from healthy donors were stored in 100% plasma with or without agitation (at 22°C or 4°C, respectively) at a concentration of 3x1011/L and sampled on days 0, 3, 7, and 14. Microparticles were detected by flow cytometry as described previously. For proteomic analysis, platelets were washed and digested with trypsin. Tryptic peptides were analyzed by nanoflow liquid chromatography electrospray ionization tandem mass spectrometry (nano LC-MS/MS). MS/MS spectra were searched against the human protein database using Proteome Discoverer 2.4 software. A student t-test test was used to determine significant differences in analytes amongst the different storage groups. Results/Finding: Under both storage conditions, GPIbα and GPV decreased significantly over storage time. However, comparison of the decline in these proteins to GPIbβ, GPIX, and other membrane proteins indicated that the mechanisms for this decline differ in the two conditions. At RT, the decrease in GPIbα and GPV appears to be largely proteolytic, given that only a minor concomitant decrease in surface level was seen in the protease-insensitive GPIX and a slight increase in GPIbβ. In addition, a comparable decrease in GPIbα level was not observed when a cytoplasmic GPIbα peptide was assayed, suggesting the extracellular portion had been proteolytically removed. In contrast, at 4°C the decline in GPIbα and GPV was accompanied by a modest decrease in GPIX, and only a small decrease in the ratio of extracellular to cytoplasmic to GPIbα peptide. These results suggested that, at 4°C, in addition to proteolysis, which was attenuated as compared to RT storage, another mechanism was responsible for removal of full-length GPIbα and other polypeptides. One such mechanism that could explain this would be loss of membrane from the platelets during 4°C storage. Indeed, we found that extracellular vesicles accumulated in the platelet supernatant during 4°C to a much higher level than at RT storage. Summary: One of the hallmarks of the platelet storage lesion at RT is shedding of surface membrane proteins including GPIbα. Previous studies in stored mouse and human platelets revealed a cleavage mechanism dependent on the metalloproteinase ADAM17. However, whether GPIbα is lost by the same mechanism in cold-stored human platelets was unknown. Our targeted proteomics analysis confirms that proteolysis is a major cause of GPIbα loss at RT, but is a less prominent mechanism at 4°C. However, another mechanism for membrane protein loss is more prominent at the lower temperature: microvesiculation. Thus, these studies provide new insights into the platelet storage lesion and suggest that measures to prevent them will have to be tailored to the dominant mechanism operating at a particular storage temperature. Disclosures No relevant conflicts of interest to declare.

Background: Current blood banking practices dictate that platelets can only be stored at room temperature (RT) for up to seven days because of functional decline and risk of bacterial overgrowth. A lower storage temperature has the potential to slow platelet metabolism and reduce oxidative damage during storage. In the current study, we quantified by mass spectrometry small molecule thiols and disulfides as markers of oxidative stress in platelets stored in plasma at RT or 4°C. Because glutathione (GSH), γGlu-Cys-Gly, is the major thiol in platelets, we also analyzed metabolites (amino acids and dipeptides) involved in GSH synthesis and degradation cycles. Method: Platelets were acquired from five healthy donors and adjusted to 3 × 1011/L in plasma. The platelets from each donor were split into two bags and stored in plasma at RT or 4°C (with or without agitation, respectively) for up to 14 days. Aliquots from each group were taken at days 0, 3, 7, and 14, and N-ethylamaleimide (NEM) was added to block free thiols before storing the samples at -80°C until analysis. Total and reduced forms of GSH and cysteine including protein-bound disulfides were quantified by mass spectrometry as described previously [Fu X. et al. Scientific Reports. 2019; 9(115):1-9]. Paired t-tests were used to evaluate the differences between the groups. Results: GSH is the major intracellular antioxidant that maintains a reducing environment within cells. After 7 days of storage at RT, reduced GSH decreased drastically from 13.0±1.2 pre-storage to 1.7±0.6 µM (p <0.005), an 85% decrease. In contrast, GSH in platelets stored at 4°C decreased at a much slower rate, with over 60% and 50% GSH remaining after 7 and 14 days of storage, respectively, indicating that platelets stored at 4°C maintain higher reducing capacities than those stored at RT. The decrease in reduced GSH can be caused by oxidation and/or degradation. If it is primarily due to oxidation, total GSH ― the sum of reduced and oxidized (disulfides) forms ― should remain relatively constant. The total GSH in the stored platelets decreased in a manner similar to the reduced GSH at both RT and 4°C, suggesting that degradation, not oxidation, was responsible for the decrease in reduced GSH. We then determined the quantities of metabolites related to GSH synthesis and degradation and found significant increases in the total amounts of the amino acids Glu, Cys, and Gly, suggesting that more GSH was degraded than synthesized in platelets during storage. We also examined the changes of Cys during storage at RT and 4°C because Cys is the main thiol in plasma and the platelets were stored in 100% plasma. Protein-bound Cys was significantly elevated in the samples stored at RT compared to those stored at 4°C, the differences being observed as early as day 3 and continuing to day 14 day (p<0.01). We previously identified protein-bound Cys as a marker for oxidative stress in plasma [Fu X. et al. Scientific Reports. 2019; 9(115):1-9]; its elevation is therefore indicative of increased oxidation of the platelet sample during RT storage compared to 4°C storage. Summary: In this study, we found that the ability of platelets to withstand oxidative stress during storage is severely compromised in platelets stored at RT as opposed to 4°C. This storage lesion affected both intracellular (GSH) and extracellular (Cys) antioxidant molecules and involved 2 mechanisms: GSH degradation and Cys oxidation. We are currently attempting to determine whether the differences observed result from increased metabolic rate in the RT-stored platelets or from greater exposure to ambient air caused by the need for agitation. These studies will identify targets for improving the quality and shelf-life of platelets stored for transfusion. Disclosures No relevant conflicts of interest to declare.

LC-MS/MS with multiple reaction monitoring (MRM) is a powerful tool for targeted metabolomics analysis including screening and quantification of known metabolites. Given the complexity of biological samples, the difference in ionization efficiency, and signal intensity of each metabolite, isotopically labeled internal standards are often used for accurate quantification. In this chapter, we describe a detailed protocol for the quantitative analysis of polyunsaturated fatty acids (PUFAs) and their oxidized products (oxylipins) by LC-MS/MS-MRM with isotope dilution. PUFAs are very susceptible to oxidation by both enzymatic and nonenzymatic pathways. Free PUFAs and corresponding oxylipins, known as bioactive lipids, are involved in many processes with varying biological functions depending on their chemical structure and concentration. Accurate quantification is thus becoming crucial to understanding the role of these bioactive lipids in health, disease(s), and other settings.